The long-term objective of this research proposal is to elucidate the molecular mechanisms underlying the glucose signal transduction pathway in plants using Arabidopsis thaliana as a model system. Sugar production through photosynthesis is the most important activity in plants and supports all life on the planet. Recent studies from my laboratory have provided compelling evidence to support the new concept that sugars are acting as central signaling molecules to modulate photosynthetic gene expression and coordinate many vital processes, including germination, seedling development, leaf and root differentiation, flowering, and senescence in plants. By using mutants, transient expression, and transgenic plants, we have provided new evidence that glucose signaling can be uncoupled from glucose metabolism, and at least one specific hexokinase (AtHXK1) has dual catalytic and regulatory roles as a major glucose sensor/signal transducer in plants. The proposed studies employing a plant model A.thaliana will reveal unconventional and exciting new insights into the molecular mechanisms of glucose sensing and signaling in plants with potential implications in other eukaryotes. A combination of molecular, biochemical, genetic, and cellular approaches will be used. Three specific aims are: 1) Define the glucose responses mediated through AtHXK1 as a sensor/signal transducer An Arabidopsis AtHXK1 knockout mutant and transgenic plants with altered HXK levels will be used to identify HXK- mediated glucose-responsive target genes. A comprehensive sets of glucose-dependent bioassays will be tested to define HXK- mediated developmental activities. Glucose phosphorylation and accumulation will be monitored. These experiments will establish the standards for analyzing sugar responses in plants and will unequivocally define the role of specific HXK as a glucose sensor/signal transducer by genetic means. 2) Elucidate the molecular mechanism of AtHXK1 function in glucose sensing and signaling Comprehensive experiments are proposed to determine the expression patterns and subcellular localization of AtHXK1, establish AtHXK1 functional assays, generate and analyze AtHXK1 mutants, characterize the AtHXK1 protein complex, and identify AtHXK1 signaling partners. 3) Identify the components in the glucose signal transduction pathways The Arabidopsis mutants that are glucose insensitive (gin) will be phenotypically and molecularly characterized. The gene for the most interesting mutant gin1 will be isolated and analyzed.